Hedyotis diffusa extract as well as preparation method and application thereof

Abstract

The invention provides a hedyotis diffusa extract and a preparation method thereof, the hedyotis diffusa extract is prepared by taking a hedyotis diffusa medicinal material as a raw material, adding a 75-85% ethanol solution for extraction, and filtering; concentrating the filtrate until no alcohol smell exists, precipitating with water, and filtering; separating the filtrate by using macroporous resin, sequentially adding 1.5-2.5 BV of water and 3-4BV of 55-65% ethanol solution for elution, collecting 2BV-3BV sections at the tail end of ethanol eluent, and concentrating to obtain a finished product. The invention further provides application of the oldenlandia diffusa extract in preparation of drugs for preventing and / or treating acute gastric mucosal lesion, gastric ulcer and reflux esophagitis, and particularly, the acute gastric mucosal lesion caused by alcohol or acute alcoholic gastric mucosal lesion and the gastric ulcer caused by excessive gastric secretion are included. In addition, the invention also provides a preparation containing the oldenlandia diffusa extract. The oldenlandia diffusa extract provided by the invention has a better curative effect of resisting acute gastric mucosal lesion, the preparation process is green and environment-friendly, the preparation method is simple, and the oldenlandia diffusa extract is suitable for industrial popularization.

Description

Technical Field

[0001] This invention belongs to the field of plant extracts, and specifically relates to an extract of Hedyotis diffusa, its preparation method, and its uses. Background Technology

[0002] Hedyotis diffusa willd., a plant belonging to the Rubiaceae family and the Hedyotis genus, has the effects of clearing heat, promoting diuresis, and detoxifying. Modern pharmacological studies have shown that the whole plant of Hedyotis diffusa contains a variety of medicinal components, including flavonoids, anthraquinones, iridoid glycosides, sterols, polysaccharides, and phenolic acids, which have anti-tumor, anti-inflammatory, antioxidant, and immunomodulatory effects.

[0003] The article "An Overview of the Clinical Applications of Hedyotis diffusa" (Straits Pharmaceutical Journal, 2008(12):99-101) discloses that Hedyotis diffusa has antioxidant and gastric mucosal protective effects, repairs gastric mucosal histological changes, inhibits gastric mucosal dysplasia, and has a certain inhibitory effect on Helicobacter pylori. Therefore, it can be used to treat acute and chronic gastritis, erosive gastritis, superficial gastritis, atrophic gastritis, and other diseases. Clinically, Hedyotis diffusa is used in combination with Artemisia capillaris, Bupleurum chinense, Citrus aurantium, Atractylodes macrocephala, and Forsythia suspensa to treat chronic gastritis.

[0004] The aqueous decoction and alcohol precipitation extract of Hedyotis diffusa can protect against indomethacin-induced gastric mucosal damage in rats. The study "Protective Effect of Hedyotis diffusa on Indomethacin-Induced Gastric Mucosal Damage in Rats" (Hebei Journal of Traditional Chinese Medicine, 2001, 23(1):70-71) disclosed that the aqueous decoction and alcohol precipitation extract of Hedyotis diffusa (concentration of 1g of crude drug per mL of decoction) can significantly increase the activity of superoxide dismutase (SOD) in serum and gastric tissue of rats with gastric ulcers induced by indomethacin and reduce the content of malondialdehyde (MDA), suggesting that its mechanism of action is related to its antioxidant effect.

[0005] Furthermore, the methanol extract of *Hedyotis puberula*, a different species within the same genus as *Hedyotis diffusa*, possesses gastric protective potential. The study "Protective effects of methanol extract of *Hedyotis puberula* (G. Don) R. Br. ex Arn. against experimentally induced gastric ulcers in rat" (J Ethnopharmacol, 2010, 131(1):216-219) investigated the gastric protective potential of the methanol extract of *Hedyotis puberula* in rat models of gastric ulcers induced by indomethacin, ethanol, and pyloric ligation. Oral administration of 400 mg / kg of *Hedyotis puberula* extract protected rats from ulcer damage, with efficacy comparable to the reference drug omeprazole. This extract protected rats from the development of gastric lesions by increasing pH and decreasing the volume, acidity, and pepsin content of gastric secretions. However, even within the same genus, the active ingredients differ between different species. The study, "Evaluation of the Quality of Hedyotis corymbosa Based on HPLC Fingerprinting Combined with Chemical Pattern Recognition" (Chinese Journal of Modern Applied Pharmacy, 2025, 42(09):1-10), showed through fingerprint analysis that compared with Hedyotis corymbosa, Hedyotis corymbosa had 9 fewer peaks (3-5, 7-12), and Hedyotis tenellifloa had 6 fewer peaks (4-5, 9-12). The similarity between the fingerprints of Hedyotis corymbosa, Hedyotis tenellifloa, and Hedyotis corymbosa ranged from 0.507 to 0.683, indicating significant differences in chemical composition between these two plants and Hedyotis corymbosa. Furthermore, Hedyotis corymbosa is often used as a counterfeit of Hedyotis corymbosa. Various methods for identifying Hedyotis corymbosa and Hedyotis corymbosa have been developed, such as morphological identification, molecular methods, microscopic identification, thin-layer chromatography, and HPLC fingerprinting. It is evident that different plants of the same genus have significantly different components and uses, and cannot be used interchangeably.

[0006] The existing technology discloses that the extract of Hedyotis diffusa is obtained by simple water extraction and alcohol precipitation, without further enriching the effective components for anti-acute gastric mucosal damage and gastric ulcer, and the dosage is relatively large; moreover, the existing technology does not disclose the application of Hedyotis diffusa extract for reflux esophagitis. Summary of the Invention

[0007] This invention provides an extract of Hedyotis diffusa, which, by enriching the effective components for treating acute gastric mucosal damage and gastric ulcers, solves the problem of large dosage in existing technologies; at the same time, the extract of Hedyotis diffusa provided by this invention can also be used for the indication of reflux esophagitis to relieve symptoms of excessive gastric acid secretion.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0009] The first objective of this invention is to provide an extract of Hedyotis diffusa, the preparation method of which includes the following steps:

[0010] (1) Take the herbal medicine of Hedyotis diffusa, add 75% to 85% ethanol solution to extract it, and filter it;

[0011] (2) Concentrate the filtrate until there is no alcohol odor, allow it to settle in water, and then filter.

[0012] (3) Separate the filtrate through macroporous resin, add 1.5 BV to 2.5 BV of water and 3 BV to 4 BV of 55% to 65% ethanol solution sequentially for elution, collect the last 2 BV to 3 BV of ethanol eluent, concentrate and dry to obtain the final product.

[0013] Furthermore, in step (1), the amount of ethanol solution used for extraction is 10 to 20 times the amount of medicinal material fed in, and the extraction method is heating and reflux extraction.

[0014] Furthermore, in step (2), water precipitation involves adding 2.5 to 3.5 times the amount of medicinal materials to water and letting it stand overnight.

[0015] Furthermore, in step (3), the macroporous resin is selected from weakly polar macroporous adsorption resins, and the weakly polar macroporous adsorption resin model is selected from AB-8, X-5, HPD-450, HPD-722, DM130, HC-200S.

[0016] The second objective of this invention is to provide a method for preparing the above-mentioned extract of Hedyotis diffusa, specifically including the following steps:

[0017] (1) Take the herbal medicine of Hedyotis diffusa, add 75% to 85% ethanol solution to extract it, and filter it;

[0018] (2) Concentrate the filtrate until there is no alcohol odor, allow it to settle in water, and then filter.

[0019] (3) Separate the filtrate through macroporous resin, add 1.5 BV to 2.5 BV of water and 3 BV to 4 BV of 55% to 65% ethanol solution sequentially for elution, collect the last 2 BV to 3 BV of ethanol eluent, concentrate and dry to obtain the final product.

[0020] A third objective of this invention is to provide the use of the above-mentioned Hedyotis diffusa extract in the preparation of remedies for the prevention and / or treatment of acute gastric mucosal injury, gastric ulcer, and reflux esophagitis.

[0021] Furthermore, acute gastric mucosal injury refers to alcohol-induced acute gastric mucosal injury or acute alcoholic gastric mucosal injury; gastric ulcer is a gastric ulcer caused by excessive gastric acid secretion.

[0022] Furthermore, the medication is one that reduces the amount of gastric juice secretion.

[0023] A fourth objective of this invention is to provide a formulation comprising the above-mentioned Hedyotis diffusa extract, wherein the Hedyotis diffusa extract is the active ingredient.

[0024] The active ingredients of *Hedyotis diffusa* include terpenes, polysaccharides, flavonoids, and polyphenols, which have certain pharmacological effects in anti-tumor, antioxidant, anti-inflammatory, and immunomodulatory aspects. Existing technologies indicate that anti-oxidative stress is the mechanism of action of *Hedyotis diffusa* in treating gastric mucosa. This invention uses a specific preparation process, employing alcohol extraction and water precipitation, followed by macroporous resin column chromatography to enrich the effective components and obtain *Hedyotis diffusa* extract. Pharmacological tests show that the extract obtained by this invention has lower total polysaccharide and total flavonoid content, and while the total terpene content is similar, it exhibits lower antioxidant stress capacity but better therapeutic effect against acute gastric mucosal injury. This breaks the technical bias that higher content of effective ingredients equates to better efficacy, producing unexpected technical results.

[0025] Furthermore, the Hedyotis diffusa extract provided by this invention can also reduce gastric acid secretion, treating gastric ulcers and reflux esophagitis caused by excessive gastric acid secretion. The pyloric ligation rat model is an animal model widely used in gastric ulcer research. By surgically ligating the pylorus of rats, gastric emptying obstruction is simulated, leading to increased gastric pressure, gastric acid retention, and consequently, gastric mucosal damage and ulcer formation. Moreover, pyloric ligation is also a commonly used method for preparing rat models of reflux esophagitis. The Hedyotis diffusa extract provided by this invention can effectively reduce gastric acid secretion in the pyloric ligation rat model and can be used to treat or alleviate symptoms of gastric ulcers and reflux esophagitis.

[0026] This invention has the following advantages:

[0027] (1) The extract of Hedyotis diffusa provided by the present invention has a better therapeutic effect on acute gastric mucosal injury.

[0028] Ethanol-induced gastric ulcer models are widely used to study the protective effects of drugs against acute gastric mucosal injury. The mechanism by which ethanol affects ulcer formation is that high concentrations of ethanol can directly damage gastric mucosal tissue, causing acute mucosal inflammation, congestion, edema, bleeding, erosion, and gastric mucosal ulceration.

[0029] Analysis of the component content and gastric mucosal damage inhibition rate of extracts 5-7 in the control group revealed that higher contents of total polysaccharides, total flavonoids, and total terpenes resulted in better damage inhibition rates, indicating that these components are effective in treating gastric mucosal damage. As shown in Table 1, the total polysaccharide, total flavonoid, and total terpenes contents of extract 2 from *Hedyotis diffusa* provided by this invention are all higher than those of extract 5 obtained by water extraction and alcohol precipitation in the prior art. Furthermore, extract 2 exhibits a gastric mucosal damage inhibition rate of 44.26% at a dose of 10g crude drug / kg, significantly higher than the 3.26% inhibition rate of extract 5 at a dose of 20g crude drug / kg. In addition, extract 2 shows a higher gastric mucosal damage inhibition rate at a dose of 10g / kg than extract 7 obtained by 80% ethanol extraction, requiring a lower dosage. The *Hedyotis diffusa* extract provided by this invention can better concentrate the active ingredients against gastric mucosal damage, improve therapeutic efficacy, and effectively reduce drug dosage.

[0030] Compared with the control group extract 4, extract 2 had lower total polysaccharide and total flavonoid content, while the total terpene content was similar. Extract 2 exhibited an inhibition rate of more than 13 times that of extract 4 against alcohol-induced gastric mucosal damage at a dose of 10 g / kg, and this difference was statistically significant compared to the model group (P<0.01). Therefore, the medicinal components of *Hedyotis diffusa* obtained by this invention through a specific process break the conventional thinking that higher concentrations of enriched medicinal components equate to better efficacy, and yield a *Hedyotis diffusa* extract with superior anti-acute gastric mucosal damage effects.

[0031] Table 1. Component content and inhibitory effect of extracts 2, 4-7 on gastric mucosal damage.

[0032]

[0033]

[0034] Note: Compared with the model group, ** indicates P<0.01.

[0035] Glutathione (GSH) and superoxide dismutase (SOD) can rapidly convert superoxide free radicals into safe, inactive biomolecules, reducing lipid peroxidation. MDA is the final product of polyunsaturated fatty acid peroxidation in cell membranes and is commonly used as a reliable marker of lipid peroxidation in tissues. As shown in Table 2, the serum GSH content in the model group rats was increased, SOD activity was decreased, and the content of lipid peroxides (MDA) was increased, indicating that the body was in a state of antioxidant system imbalance. Extract 2 had a lower ability to inhibit GSH content than extract 4; its effect on promoting SOD activity was lower than that of extracts 6-7; and its ability to inhibit MDA content was lower than that of extract 6. The antioxidant stress test results of extracts 2 and 4-7 proved that the antioxidant stress capacity of extract 2 provided by this invention was lower than that of extracts 4-7.

[0036] Existing technologies indicate that anti-oxidative stress is the mechanism of action of Hedyotis diffusa in treating gastric ulcers. This invention does not improve the gastric mucosal damage inhibition rate by enriching anti-oxidative stress components, but rather uses a specific preparation process to enrich the active ingredients. Through the specific relationship between the preparation process, components, and efficacy, a better therapeutic effect against alcohol-induced acute gastric ulcers is achieved, producing unexpected technical results.

[0037] Table 2. Effects of extracts 2, 4–7 on the biochemical effects of ethanol-induced gastric mucosal injury in rats.

[0038]

[0039] Note: Compared with the model group, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001.

[0040] (2) The extract of Hedyotis diffusa provided by the present invention can significantly inhibit excessive gastric juice secretion and can be used to treat reflux esophagitis and gastric ulcers caused by excessive gastric juice secretion.

[0041] This invention employs pyloric ligation to prevent normal gastric emptying, causing gastric juice to remain in the stomach. This prolongs the action time and intensifies the effect of gastric acid and pepsin within the stomach. Simultaneously, pyloric ligation leads to antral expansion. This expansion stimulates G cells in the antral region to secrete gastrin via nerve reflexes. Gastrin acts on the gastric parietal cells, causing excessive gastric juice secretion, thus mimicking the pathological states of gastric ulcers and reflux esophagitis caused by excessive gastric juice secretion. The *Hedyotis diffusa* extract 1 provided by this invention reduces gastric juice secretion by 63% compared to the model group, effectively treating and alleviating the symptoms of excessive gastric juice secretion caused by pyloric ligation. It can be used to treat reflux esophagitis and gastric ulcers caused by excessive gastric juice secretion.

[0042] Table 3. Effects of Extract 1 on gastric secretion in rats with pyloric ligation (n=6)

[0043]

[0044] Note: Compared to the model group, * P < 0.05 ** P < 0.01.

[0045] (3) The preparation process of the white flower snake tongue extract provided by the present invention is green and environmentally friendly, the preparation method is simple, and it is suitable for industrial promotion. Attached Figure Description

[0046] Figure 1A schematic diagram of ethanol-induced gastric mucosal injury modeling and administration in rats. The extract of Hedyotis diffusa was administered by gavage for 14 consecutive days. From day 13 to 15, the rats were fasted but allowed free water for 24 hours. After the fasting period, the rats were administered anhydrous ethanol by gavage and then allowed free water for 1 hour. The rats were then sacrificed and their tissues were collected.

[0047] Figure 2 Hepatic histopathological section of ethanol-induced gastric mucosal damage in rats induced by extracts of Hedyotis diffusa.

[0048] Figure 3 A schematic diagram of pyloric ligation-induced gastric mucosal injury modeling and drug administration. The rats were continuously administered Hedyotis diffusa extract by gavage for 7 days. From day 6 to 8, the rats were fasted but allowed free water for 24 hours. After the fasting period, the rats underwent laparotomy under isoflurane anesthesia. The abdomen was disinfected, and the abdominal cavity was opened below the xiphoid process. The stomach was gently pulled out of the abdominal cavity to expose the pylorus. The junction of the pylorus and duodenum was ligated with surgical sutures, and the incision was sutured. After a strict 4-hour fast, the rats were sacrificed, the stomach was removed, and the gastric fluid was collected. Detailed Implementation

[0049] The following embodiments are intended to further illustrate the present invention, but the scope of protection of the present invention is not limited to the following embodiments. The ethanol eluent collection points are divided according to the eluent outflow time; the first to flow out is called the eluent front, and the second to flow out is called the eluent end. Each column volume segment corresponds to the collected eluent volume. The 1 BV segment at the end of the eluent represents the 1 BV eluent volume before the eluent flow stops. The 0.5 BV-1.5 BV segment at the front of the eluent represents the 0.5 BV-1.5 BV eluent volume from the start of outflow.

[0050] Example 1: Preparation of Hedyotis diffusa extract 1

[0051] Take 500g of Hedyotis diffusa herb, add 20 times the amount of herb to 75% ethanol solution, heat and reflux to extract three times, one hour each time, and filter. Combine the filtrates and concentrate until there is no alcohol taste. Add 2.5 times the amount of herb to water, let stand overnight, and filter. Pass the filtrate through an X-5 macroporous resin column, add 1.5 BV of water and 3 BV of 55% ethanol solution to elute sequentially, collect the last 2 BV segment of the ethanol eluent, concentrate, and dry to obtain the final product.

[0052] Example 2: Preparation of Hedyotis diffusa extract 2

[0053] Take 2 kg of Hedyotis diffusa herb, add 15 times the amount of herb to 80% ethanol solution, heat and reflux twice for 2 hours each time, and filter. Combine the filtrates and concentrate until there is no alcohol taste. Add 3 times the amount of herb to water, let stand overnight, and filter. Pass the filtrate through an AB-8 macroporous resin column, add 2 BV of water and 3.5 BV of 60% ethanol solution to elute sequentially, collect the last 2.5 BV segment of the ethanol eluent, concentrate, and dry to obtain the final product.

[0054] Example 3: Preparation of Hedyotis diffusa extract 3

[0055] Take 3 kg of Hedyotis diffusa herb, add 10 times the amount of herb to 85% ethanol solution, heat and reflux for 1 hour, then filter. Concentrate the filtrate until there is no alcohol odor, add 3.5 times the amount of herb to water, let stand overnight, and filter. Pass the filtrate through a DM130 macroporous resin column, add 2.5 BV of water and 4 BV of 65% ethanol solution sequentially for elution, collect the last 3 BV segment of the ethanol eluent, concentrate, and dry to obtain the final product.

[0056] Example 4: Preparation of Hedyotis diffusa extract 4

[0057] Take 2 kg of Hedyotis diffusa herb, add 15 times the amount of herb to 80% ethanol solution, heat and reflux twice for 2 hours each time, and filter. Combine the filtrates and concentrate until there is no alcohol taste. Add 3 times the amount of herb to water, let stand overnight, and filter. Pass the filtrate through an AB-8 macroporous resin column, add 2 BV of water and 3.5 BV of 60% ethanol solution to elute sequentially, collect the first 0.8 to 1.2 BV of ethanol eluent, concentrate, and dry to obtain the final product.

[0058] Example 5: Preparation of Hedyotis diffusa extract 5

[0059] A water decoction and alcohol precipitation control group was set up based on existing technology to compare the effective components and efficacy.

[0060] Take 2 kg of Hedyotis diffusa herb, add 15 times the amount of herb to an 80% ethanol solution, soak for 2 hours, and filter. Add 15 times the amount of herb to water, heat and reflux twice for 2 hours each time, and filter. Combine the filtrates and concentrate to 3 times the amount of herb, and filter. While stirring, add ethanol to the filtrate to a concentration of 40% ethanol, filter, collect the filtrate, and wash the resulting precipitate 4 times with an 80% ethanol solution equal to the amount of herb, and collect the washing liquid. Combine the filtrate and washing liquid, concentrate, and dry to obtain the final product.

[0061] Example 6: Preparation of Hedyotis diffusa extract 6

[0062] Take 1.5 kg of Hedyotis diffusa herb, add 15 times the amount of water, heat and reflux to extract twice, 2 hours each time, filter while hot, combine the filtrates, concentrate and dry to obtain the final product.

[0063] Example 7: Preparation of Hedyotis diffusa extract 7

[0064] Take 1.5 kg of Hedyotis diffusa herb, add 15 times the amount of herb to an 80% ethanol solution, heat and reflux to extract twice, 2 hours each time, filter while hot, combine the filtrates, concentrate and dry to obtain the final product.

[0065] Example 8: Component Content Detection

[0066] The total polysaccharide, total flavonoid, and total terpene contents of the extracts of Hedyotis diffusa prepared in Examples 2, 4-7 were determined.

[0067] 1. Detection Method

[0068] 1.1 Method for determining total polysaccharide content

[0069] Preparation of reference solution: Take an appropriate amount of D-anhydrous glucose, accurately weigh it, add water to make a solution containing 90 μg per 1 mL.

[0070] Preparation of the standard curve: Accurately transfer 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the reference solution into 10 mL stoppered test tubes, add water to each to 1.0 mL, accurately add 1 mL of 5% phenol solution (prepare immediately before use), shake well, and then slowly and accurately add 5 mL of sulfuric acid along the wall, shake well, heat in a boiling water bath for 20 minutes, remove, and cool in an ice bath for 5 minutes. Using the corresponding reagent as a blank, measure the absorbance at a wavelength of 488 nm using ultraviolet-visible spectrophotometry (General Rule 0401). Plot the standard curve with absorbance as the ordinate and concentration as the abscissa.

[0071] Assay: Weigh approximately 15 mg of the extract accurately into a 100 mL volumetric flask, add water to dissolve, add water to the mark, shake well, accurately measure 1 mL, place in a 10 mL stoppered test tube, and measure the absorbance according to the method under the preparation of the standard curve, starting from "accurately add 1 mL of 5% phenol solution". Read the value of D-anhydrous glucose in the test solution from the standard curve and calculate the result.

[0072] 1.2 Method for determining total flavonoid content

[0073] Preparation of reference solution: Take an appropriate amount of rutin, accurately weigh it, and add ethanol to prepare a solution containing 0.2 mg per 1 mL.

[0074] Preparation of the standard curve: Accurately transfer 2 mL, 3 mL, 4 mL, 5 mL, and 7 mL of the reference solution into 25 mL volumetric flasks, respectively. Add ethanol to each flask to a final volume of 7.0 mL. Add 1 mL of 5% sodium nitrite solution to each flask sequentially, shake well, and let stand for 6 minutes. Add 1 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 minutes. Add 10 mL of 4% sodium hydroxide solution, then add water to the mark and shake well. Using the corresponding reagents as blanks, detect the absorbance at 510 nm using UV-Vis spectrophotometry (General Rule 0401). Plot the standard curve with absorbance on the ordinate and reference concentration on the abscissa.

[0075] Assay: Weigh 30 mg of the extract accurately and place it in a 10 mL volumetric flask. Dissolve the extract in 60% ethanol and bring the volume to a final volume. Accurately measure 2 mL of the extract into a 25 mL volumetric flask. Following the method under the preparation of the standard curve, starting from "add ethanol to 7.0 mL", detect the sample at 510 nm. Read the amount of rutin in the test solution from the standard curve and calculate the result.

[0076] 1.3 Methods for determining total terpenoid content

[0077] Preparation of reference solution: Take an appropriate amount of oleanolic acid, accurately weigh it, and add methanol to prepare a solution containing 0.2 mg per 1 mL.

[0078] Preparation of the standard curve: Accurately transfer 0.05 mL, 0.1 mL, 0.2 mL, 0.3 mL, and 0.4 mL of oleanolic acid reference solution into 10 mL stoppered test tubes, evaporate to dryness, and cool. Accurately add 0.2 mL of freshly prepared vanillin-acetic acid solution (accurately weigh 0.5 g of vanillin into a 10 mL volumetric flask, dissolve in acetic acid, and dilute to the mark; shake well). Then accurately add 0.8 mL of perchloric acid, shake well, heat in a 70 °C water bath for 15 minutes, immediately cool in an ice-water bath for 5 minutes, remove, and accurately add 4 mL of acetic acid; shake well. Using the corresponding reagent as a blank, measure the absorbance at 546 nm using UV-Vis spectrophotometry (General Rule 0401). Plot the standard curve with absorbance as the ordinate and the concentration of the reference standard as the abscissa.

[0079] Assay: Weigh 30 mg of the extract accurately and place it in a 10 mL volumetric flask. Dissolve the extract in 80% ethanol and bring the volume to a final volume. Accurately transfer 0.1 mL of the extract into a 10 mL stoppered test tube. Starting from the point of "evaporation", repeat the same procedure to measure the absorbance. Read the content of oleanolic acid in the test solution from the standard curve and calculate the result.

[0080] 2. Test Results

[0081] The results of the tests on the extracts obtained by the different preparation methods described above are shown in Table 4.

[0082] Table 4 Results of extract content determination

[0083] Extract Name Total polysaccharide content (%) Total flavonoid content (%) Total terpenoid content (%) Extract 2 35.74 13.33 17.56 Extract 4 41.23 19.82 16.41 Extract 5 33.97 4.68 5.46 Extract 6 43.58 5.2 7.12 Extract 7 45.94 8.79 12.34

[0084] The difference between extract 2 and extract 4 lies in the different eluent fractions collected after elution with 60% ethanol solution. Extract 4 has higher total polysaccharide and total flavonoid content than extract 2, while the total terpene content of the two is similar.

[0085] The difference between Extract 2 and Extract 5 is that Extract 5 omits macroporous resin column chromatography and mainly collects less polar components by soaking in alcohol solution and water extraction followed by alcohol precipitation. The contents of total polysaccharides, total flavonoids and total terpenoids in Extract 5 are lower than those in Extract 2.

[0086] Extract 6 and Extract 7 are water extract and alcohol extract respectively. The corresponding contents of total flavonoids and total terpenoids are significantly lower than those in Extract 2, but the total polysaccharide content is significantly higher than that in Extract 2, indicating that simple extraction is difficult to fully obtain flavonoids and terpenoid components in Hedyotis diffusa.

[0087] Example 9: Protective effect of Hedyotis diffusa extract on alcohol-induced gastric mucosal injury model in rats

[0088] 1. Experimental animals and materials

[0089] 1.1 Experimental animals

[0090] Healthy SD rats of SPF grade, weighing 250 g ± 10 g at the start of the experiment, were purchased from Shanghai Slake Animal Technology Co., Ltd., and the production license number of experimental animals is: SCXK(Shanghai)-2022-0004. Conditions for grouped feeding of animals: room temperature (24 ± 2)°C, relative humidity about 50%, artificially maintained 12-hour light / dark cycle, bedding replaced regularly, animal house kept clean and ventilated, animals could eat and drink freely, and dying and fighting animals were fed separately. All animals were adaptively fed for one week before the experiment.

[0091] 1.2 Experimental materials

[0092] Table 5 Experimental materials for alcohol-induced gastric mucosal injury in rats

[0093] name batch number Manufacturer / Source Extract 2 20241104-02 self made Extract 4 20241104-01 self made Extract 5 20241104-03 self made Extract 6 20240815-02 self made Extract 7 20240815-03 self made Hericium erinaceus extract 240901 Zhejiang Kang Enbei Traditional Chinese Medicine Co., Ltd. Total Superoxide Dismutase (T-SOD) Assay Kit A001-3 Nanjing Jiancheng Biotechnology Research Institute Reduced glutathione (GSH) assay kit A006-2-1 Nanjing Jiancheng Biotechnology Research Institute Lipid oxidation (MDA) detection kit A003-1-2 Nanjing Jiancheng Biotechnology Research Institute RatIL-6UncoatedElisaKitwithPlates 375907-001 Thermo Fisher Scientific

[0094] 1.3 Experimental group settings

[0095] Table 6 Group settings of Hedyotis diffusa extract

[0096]

[0097]

[0098] Positive control: 246 mg / mL Hericium erinaceus extract. Preparation method: Take 100 mL of Hericium erinaceus extract and mix it with 103 mL of pure water.

[0099] Negative control: 2% maltodextrin. Preparation method: Take 2 g of maltodextrin and mix it with 100 mL of pure water.

[0100] 2. Experimental methods

[0101] 2.1 Administration and modeling methods

[0102] Healthy SD rats weighing 250±10g were randomly divided into 14 groups (n=6): normal group, model group, negative control group (2% maltodextrin), positive control group (246mg / mL Hericium erinaceus extract), and extract groups of Hedyotis diffusa: extract 2 (10g / kg, 20g / kg), extract 4 (10g / kg, 20g / kg), extract 5 (10g / kg, 20g / kg), extract 6 (10g / kg, 20g / kg), and extract 7 (10g / kg, 20g / kg). Rats in each group were weighed daily, and the gavage dose was adjusted at 10mL / kg based on daily weight changes.

[0103] The treatment groups were administered the corresponding dose of drug via gavage, while the normal and model groups were administered the corresponding volume of drinking water via gavage for 14 days. From day 13 to day 15 of drug administration, all rats in the experimental groups were strictly fasted but allowed free water for 24 hours. After the fasting period, except for the normal group, all other rats were given 1 mL of anhydrous ethanol followed by 1 hour of water restriction. The rats were then sacrificed and their tissues were collected. A schematic diagram of ethanol-induced gastric mucosal injury modeling and drug administration in rats is shown below. Figure 1 As shown.

[0104] 2.2 Observation Indicators

[0105] 1) General condition observation: Record the rat's weight during the administration period, observe the color of the rat's hair, and observe the characteristics, color, and texture of the rat's stool.

[0106] 2) Visual observation and scoring: The length and width of the bleeding point or bleeding band in the gastric mucosa are measured visually using calipers. Since the width represents a much greater degree of injury severity than the length, it is scored twice. The scoring criteria are shown in Table 7.

[0107] Table 7. Scoring Rules for Gastric Mucosal Injury

[0108]

[0109] The degree of gastric mucosal damage in each experimental group was expressed as the damage score index and the damage inhibition rate (%). Damage score index = total damage score of the group / number of animals in the group; damage inhibition rate (%) = (AB) / A × 100% (A and B are the damage scores of the model group and the experimental group, respectively).

[0110] 3) Pathological Histological Observation and Scoring: After gross examination, the most severely damaged part of the gastric mucosa of each animal was excised, fixed in 4% paraformaldehyde solution, routinely prepared, and subjected to HE staining analysis for histopathological examination under a microscope. It is important to select a transverse section of the gastric mucosa, including the entire mucosal layer, for observation. The HE staining scoring method is as follows: the degree of involvement of congestion, hemorrhage, and mucosal cell degeneration and necrosis in the entire mucosal epithelial layer is divided into 5 grades. Congestion has a weight of 1, hemorrhage a weight of 2, and epithelial cell degeneration and necrosis a weight of 3. The scoring criteria and the formula for the total lesion score are shown in Table 8.

[0111] Table 8. Scoring Rules for Pathological Tissues

[0112]

[0113] 2.3 Statistical Methods

[0114] GraphPadPrism 10.4 software was used for data analysis. First, it was determined whether the data followed a normal distribution. A One-Way Anova test was used if it did, and a non-parametric test was used otherwise. Results are expressed as mean ± standard error (Mean ± SEM). A p-value < 0.05 was considered statistically significant.

[0115] 3. Test Results

[0116] 3.1 General Condition Observation

[0117] During the administration of the drugs via gavage, all rats grew normally without any abnormal behavior or death. There was no significant difference in weight gain among the groups. During the administration period, the rats in all groups had normal feces without soft stools or diarrhea, indicating that the drugs had no effect on the basic growth and development of the animals.

[0118] 3.2 Macroscopic observation and damage assessment of rat gastric tissue

[0119] Ethanol, as an inducer of gastric mucosal injury, can produce erosions, ulcers, and petechial hemorrhages on the gastric mucosa. Ethanol-induced gastric ulcer models are widely used to study the protective effects of drugs on the gastric mucosa. Macroscopic observation revealed that, compared with the normal group, rats treated with ethanol exhibited significant structural pathological changes in gastric tissue, such as submucosal hemorrhage and edema, and thin, elongated hemorrhagic bands parallel to the long axis of the stomach. The experimental results showed that Hericium erinaceus extract, 10 g / kg extract 2, and 20 g / kg extract 7 significantly reduced the ethanol-induced gastric mucosal injury index; the damage inhibition rate of 10 g / kg extract 2 was 44.26%, which was significantly higher than the damage inhibition rates of 20 g / kg extract 7 (38.91%) and Hericium erinaceus extract (38.12%), and significantly different from the model group (P<0.01).

[0120] As shown in Tables 4 and 9, the component content and damage inhibition rate of extracts 5-7 in the control group indicate that higher contents of total polysaccharides, total flavonoids, and total terpenes correlate with better damage inhibition rates, suggesting that these components are effective for treating gastric mucosal damage. The total polysaccharide, total flavonoid, and total terpenes contents of extract 2 from *Hedyotis diffusa* extracted in this invention are higher than those of extract 5 obtained by water extraction and alcohol precipitation in the prior art. Furthermore, extract 2 exhibits a gastric mucosal damage inhibition rate of 44.26% at a dose of 10 g / kg, significantly higher than the 3.26% inhibition rate of extract 5 at a dose of 20 g / kg. In addition, extract 2 shows a higher gastric mucosal damage inhibition rate at a dose of 10 g / kg than extract 7 obtained by 80% ethanol extraction, requiring a lower dosage. Therefore, the *Hedyotis diffusa* extract provided by this invention can better concentrate the active ingredients for anti-gastric mucosal damage, improve therapeutic efficacy, and effectively reduce dosage.

[0121] Compared with the control group extract 4, the extract of *Hedyotis diffusa* 2 obtained in this invention has lower total polysaccharide and total flavonoid content, while the total terpene content is similar. At a dose of 10 g / kg, the inhibition rate of alcohol-induced gastric mucosal damage is more than 13 times that of extract 4, and there is a significant difference compared with the model group (P<0.01). Therefore, the effective components obtained by this invention through a specific process break the conventional thinking that higher levels of enriched active ingredients equate to better efficacy, and yield a *Hedyotis diffusa* extract with better anti-acute gastric mucosal damage effects.

[0122] Table 9. Effects of Hedyotis diffusa extract on ethanol-induced gastric mucosal damage in rats (n=6)

[0123]

[0124] Note: Compared with the model group, * indicates P<0.05, and ** indicates P<0.01.

[0125] 3.3 Pathological observation of rat gastric tissue

[0126] HE staining results are as follows Figure 2 As shown in the figure. Compared with the normal group, the model group showed obvious pathological features such as loss of mucosal epithelial cells, bleeding band damage, submucosal edema, and significant absence of inflammatory cell infiltration glands. In rat gastric tissue pretreated with Hericium erinaceus extract, only slight damage to the superficial epithelium was observed, and submucosal edema and inflammatory cell infiltration were effectively improved. Treatment with 10 g / kg extract 2 and 10 g / kg and 20 g / kg extract 7 resulted in moderate damage to the surface epithelium of rat gastric tissue accompanied by submucosal edema and inflammatory cell infiltration. Treatment with 20 g / kg extract 2 showed damage to the surface epithelium and mucosa of rat gastric tissue, with edema and submucosal inflammatory cell infiltration, showing only improvement compared to the model group. Treatment with 2% maltodextrin showed no significant improvement compared to the model group.

[0127] As shown in Table 10, compared with the model group, the HE scores of Hericium erinaceus extract, 10 g / kg extract 2, and 10 g / kg extract 7 were significantly lower (P<0.0001, P<0.001, P<0.01). However, except for Hericium erinaceus extract, the other groups showed improvement in submucosal edema thickness, but the differences were not statistically significant.

[0128] Table 10. Effects of Hedyotis diffusa extract on ethanol-induced gastric mucosal damage in rats (HE staining)

[0129]

[0130]

[0131] Note: Compared with the model, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001.

[0132] 3.4 Biochemical and inflammatory factor detection

[0133] Oxidative stress, caused by an imbalance between oxidation levels and antioxidant defense, is a crucial pathogenic mechanism of gastric ulcers. Ethanol can also induce oxidative stress by increasing the production of reactive oxygen species and depleting cellular antioxidant capacity. MDA, the final product of polyunsaturated fatty acid peroxidation in cell membranes, is commonly used as a reliable marker of lipid peroxidation in tissues. GSH is an important cellular antioxidant involved in various cellular functions, such as the regulation of cell signaling and DNA repair mechanisms. Furthermore, studies have shown that in ethanol-induced gastric injury, SOD levels in gastric mucosa tissue are decreased, reducing the body's ability to resist oxidative stress; increasing SOD levels helps to combat oxidative stress damage.

[0134] The relevant oxidative factors were detected, and the results are shown in Table 11. After ethanol treatment, the SOD activity in the serum of rats in the model group decreased, the GSH content increased significantly, and the MDA content increased. The GSH content in the serum of rats treated with Hericium erinaceus extract, at doses of 10 g / kg and 20 g / kg for extracts 2, 4, and 7, and at a dose of 20 g / kg for extracts 5 and 6, was significantly lower than that in the model group. The SOD activity in the Hericium erinaceus extract, at doses of 10 g / kg and 20 g / kg for extracts 6 and 7, at a dose of 20 g / kg for extract 2, and at a dose of 10 g / kg for extract 5 was significantly higher than that in the model group. The serum MDA content in the Hericium erinaceus extract, at doses of 10 g / kg and 20 g / kg for extract 6, was significantly lower. The inflammatory factor IL-6 in the 20 g / kg dose of extract 5 was significantly higher than that in the model group, while there were no significant differences in the other groups. There were no significant differences in any indicators between the maltodextrin group and the model group. The various extracts of Hedyotis diffusa can regulate the peroxidation state of rats induced by ethanol damage to varying degrees.

[0135] Table 11 Effects of Hedyotis diffusa extract on biochemical inflammatory factors in ethanol-induced acute gastric mucosal injury in rats (n=6)

[0136] Note: Compared with the model group, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001.

[0137] The results of oxidative factor detection for different extracts showed that the GSH and MDA indices of extracts 4-7 were even better than those of extract 2, indicating that extracts 4-7 had better antioxidant activity. Existing technologies indicate that antioxidant stress is the mechanism of action of Hedyotis diffusa in treating gastric ulcers. This invention does not improve the gastric mucosal damage inhibition rate by enriching antioxidant stress components, but rather uses a specific preparation process to enrich the active ingredients. Through the specific relationship between the preparation process, components, and efficacy, better therapeutic effects on alcohol-induced acute gastric ulcers were achieved, producing unexpected technical results.

[0138] Example 10: Study on the effect of Hedyotis diffusa extract on gastric secretion regulation in a rat model of gastric mucosal injury induced by pyloric ligation

[0139] 1. Experimental animals and materials

[0140] 1.1 Experimental Animals

[0141] Twenty-four healthy male SD rats, SPF grade, weighing 200g ± 10g, were purchased from Shanghai Silex Animal Technology Co., Ltd. Animal housing conditions: room temperature (24±2)℃, relative humidity approximately 50%, artificially maintained 12-hour light / dark cycle, regular bedding changes, clean and well-ventilated animal housing, free access to food and water, with animals that were dying or fighting kept separately. All animals were acclimatized for one week prior to the experiment.

[0142] 1.2 Test Materials

[0143] Table 12 Experimental materials for gastric mucosal injury induced by pyloric ligation

[0144] name batch number Manufacturer / Source Extract 1 20250313-01 self made Monkey mushroom extract 240901 Zhejiang Kang Enbei Traditional Chinese Medicine Co., Ltd. pH test strips (0.5-5.0) / Hangzhou Shisan Technology Co., Ltd. pH test strips (0-6.0) / Shenzhen Hongda Precision Equipment Co., Ltd. pH test strips (1.4-3.0) / Hangzhou Shisan Technology Co., Ltd.

[0145] 1.3 Experimental group setup

[0146] Table 13 Group settings for Hedyotis diffusa extract

[0147]

[0148] Positive control: 246 mg / mL Hericium erinaceus extract. Preparation method: Mix 100 mL of Hericium erinaceus extract with 103 mL of pure water.

[0149] 2. Test methods

[0150] 2.1 Modeling Method

[0151] Healthy SD rats weighing 200±10g were selected and randomly divided into a model group, a Hericium erinaceus extract group, and groups receiving 10g / kg and 20g / kg of extract 1 (n=6). The model group received an equal volume of pure water via gavage. Rats in each group were weighed daily, and the gavage dose was adjusted by 10mL / kg based on daily weight changes. Gavage was administered continuously for 7 days. From day 6 onwards, strict fasting was maintained, but water was allowed for 24 hours. After fasting, the rats were anesthetized with isoflurane and underwent laparotomy. The abdomen was disinfected, and the abdominal cavity was incised below the xiphoid process. The stomach was gently pulled out of the abdominal cavity to expose the pylorus. The junction of the pylorus and duodenum was ligated with surgical sutures, and the incision was sutured. Postoperatively, strict fasting and water restriction were maintained for 4 hours. The rats were then sacrificed, the stomach was removed, and gastric juice was collected. After centrifugation, the volume of the supernatant was measured, and the pH value of the gastric acid in the gastric juice was measured using three different precision pH test strips. Figure 3 As shown.

[0152] 2.2 Observation Indicators

[0153] Changes in gastric juice volume and pH value.

[0154] 3. Test Results

[0155] The experimental results are shown in Table 14. The gastric volume of rats treated with Hericium erinaceus extract and extract 1 was effectively inhibited, showing a significant difference compared to the model group. However, the results of gastric acid detection using three pH test strips showed that extract 1 of Hedyotis diffusa could not significantly regulate the pH of gastric acid in the gastric juice.

[0156] Table 14 Effects of Hedyotis diffusa extract on gastric secretion in rats with pyloric ligation (n=6)

[0157]

[0158] Note: Compared to the model group, * P < 0.05 ** P < 0.01

[0159] After pyloric ligation, gastric juice cannot empty normally and remains in the stomach, prolonging the action time and increasing the intensity of gastric acid and pepsin. Simultaneously, pyloric ligation causes antral dilation, which stimulates G cells in the antrum to secrete gastrin via nerve reflexes. Gastrin acts on parietal cells, causing excessive gastric juice secretion, thus mimicking the pathological state of gastric ulcers caused by excessive gastric juice secretion. Pyloric ligation is also a commonly used method for preparing animal models of reflux esophagitis. By restricting the emptying of gastric contents, pyloric ligation increases gastric pressure, causing gastric contents to reflux into the esophagus, leading to esophageal mucosal damage and the formation of reflux esophagitis.

[0160] The experimental results showed that extract 1 could significantly inhibit excessive gastric juice secretion caused by pyloric ligation at doses of 10 g / kg and 20 g / kg, reducing gastric juice secretion by 63% compared with the model group. It effectively treated and relieved the symptoms of excessive gastric juice secretion caused by pyloric ligation and can be used to treat reflux esophagitis and gastric ulcers caused by excessive gastric juice secretion.

Claims

1. An extract of Hedyotis diffusa, characterized in that, The method for preparing the extract includes the following steps: (1) Take the herbal medicine of Hedyotis diffusa, add 75% to 85% ethanol solution to extract it, and filter it; (2) Concentrate the filtrate until there is no alcohol odor, allow it to settle in water, and then filter. (3) Separate the filtrate through macroporous resin, add 1.5 BV to 2.5 BV of water and 3 BV to 4 BV of 55% to 65% ethanol solution sequentially for elution, collect the last 2 BV to 3 BV of ethanol eluent, concentrate and dry to obtain the final product.

2. The extract according to claim 1, characterized in that, The amount of ethanol solution used in step (1) is 10 to 20 times the amount of medicinal material, and the extraction method is heating and reflux extraction.

3. The extract according to claim 1, characterized in that, The step (2) water settling involves adding 2.5 to 3.5 times the amount of medicinal materials to water and letting it stand overnight.

4. The extract according to claim 1, characterized in that, The macroporous resin in step (3) is selected from weakly polar macroporous adsorption resins.

5. The extract according to claim 4, characterized in that, The weakly polar macroporous adsorption resin is selected from one of AB-8, X-5, HPD-450, HPD-722, DM130, and HC-200S.

6. A method for preparing the extract of Hedyotis diffusa as described in any one of claims 1 to 5, characterized in that, The preparation method includes the following steps: (1) Take the herbal medicine of Hedyotis diffusa, add 75% to 85% ethanol solution to extract it, and filter it; (2) Concentrate the filtrate until there is no alcohol odor, allow it to settle in water, and then filter. (3) Separate the filtrate through macroporous resin, add 1.5 BV to 2.5 BV of water and 3 BV to 4 BV of 55% to 65% ethanol solution sequentially for elution, collect the last 2 BV to 3 BV of ethanol eluent, concentrate and dry to obtain the final product.

7. The use of the extract of Hedyotis diffusa as described in any one of claims 1 to 5 in the preparation of a medicament for the prevention and / or treatment of acute gastric mucosal injury, gastric ulcer and reflux esophagitis.

8. The use according to claim 7, characterized in that, The acute gastric mucosal injury is alcohol-induced acute gastric mucosal injury or acute alcoholic gastric mucosal injury, and the gastric ulcer is a gastric ulcer caused by excessive gastric acid secretion.

9. The use according to claim 7, characterized in that, The drug in question is one that reduces the amount of gastric juice secretion.

10. A formulation comprising the extract of *Hedyotis diffusa* as described in any one of claims 1 to 5, characterized in that, The preparation uses Hedyotis diffusa extract as its active ingredient.

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